Permeability Assay Conditions

Permeability of compounds across a cell layer is measured in order to determine the absorption potential of a compound or a chemical series and select compounds for in vivo studies. Apparent permeability coefficients can be used to compare compounds within a series for ranking. Between different laboratories comparison of compounds should be done only on the basis of classification (high, medium, low) since permeability coefficients can differ between the labs (Artursson et al. 2001 see Critical Assessment of the Method). 

As experimental buffer for washing and permeability assay HBSS, pH 7.4 is proposed. 

Compound dilutions should be performed from 10 mM compound stock solutions in DMSO at the day of experiment. Compounds should be taken from micro well plates prepared by the different robot systems dilution is prepared with buffer or intermediate steps (containing DMSO) to avoid precipitation of compounds with low solubility. Final DMSO concentration should not exceed 0.5%. Alternatively, compound dilutions are prepared manually to result in maximal final concentration of DMSO of 0.5 %. 

Compound concentration should be chosen on the basis of solubility data. Compound concentrations of 50 pM should not be exceeded in routine tests. Concentrations lower than 6.25 pM should be avoided due to possible analytical limitations. 

As an example, 4 different categories of compound concentrations could be used due to differences in projects and compound characteristics 50 pM, 25 pM, 12.5 pM, 6.25 pM. 

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Sugano, K. Hamada, H. Machida, M. Ushio, H. Saitoh, K. Terada, K., Optimized conditions of biomimetic artificial membrane permeability assay, Int. J. Pharm. 228, 181-188 (2001). 

The literature survey in this section suggests that the ideal in vitro permeability assay would have pH 6.0 and 7.4 in the donor wells, with pH 7.4 in the acceptor wells. (Such a two-pH combination could differentiate acids from bases and non-ionizables by the differences between the two Pe values.) Furthermore, the acceptor side would have 3% wt/vol BSA to maintain a sink condition (or some sinkforming equivalent). The donor side may benefit from having a bile acid (i.e., taurocholic or glycocholic, 5-15 mM), to solubilize the most lipophilic sample molecules. The ideal lipid barrier would have a composition similar to those in Table 3.1, with the membrane possessing a substantial negative charge (mainly from PI). Excessive DMSO/other co-solvents would be best avoided, due to their unpredictable effects. 

Sugano K, Hamada H, Machida M, Ushio H, Saitoh K, Terada K (2001b) Optimized conditions of bio-mimetic artificial membrane permeability assay. Int J Pharm 228 181-188. 

Under these culture and assay conditions PBCEC displayed sucrose permeabilties as low as 2 x 10 7cm/s. This tightness becomes close to in vivo-permeabilities of 3 x 10 8cm/s (Ohno et al. 1978) and 1.2 x 10 7cm/s (Levin et al. 1980). Data are expressed as mean standard deviation (n = 3-6). 

Solubility measurements are made to determine an intrinsic property, which influences the absorption potential of a compound [3]. Even though solubility itself does not directly dictate the absorption of a drug, it is important to consider solubility in relation to permeability and potency. In addition, in medicinal chemistry projects, there are other issues to consider that may also be affected by poor solubility, particularly insolubility under screening assay conditions. 

Alternatively, a well-validated poorly permeable compound is included in the analyte solutions as an internal standard. A compound such as theophylline has an effective permeability of 0.12 x 10 scm/s (exact value will depend on assay conditions). If the compound were seen to be permeating significantly faster than this effective permeability, it could be concluded that the membrane had been compromised. This approach would mean that a detection method based on separation, such as HPLC, would be needed. Upon the inclusion of a second internal standard, which was known to be highly soluble, such as verapamil, effective permeability 16 x lCTscm/s would enable monitoring of the incubation time to ensure that equilibrium had not been reached for highly permeable compounds. 

These automated assays can be used for high-throughput ADME screening in early drug discovery. The double-sink PAMPA permeability assay mimics in vivo conditions by the use of a chemical sink in the acceptor wells and pH gradient in the donor wells. The use of the pION gut-box integrated on the deck has shortened the PAMPA assay incubation time to 30 minutes. The permeability coefficient and rank order correlate well with data obtained using the in vitro Caco-2 assay and in vivo permeability properties measured in rat intestinal perfusions. 

The equations used to calculate permeability coefficients depend on the design of the in vitro assay to measure the transport of molecules across membrane barriers. It is important to take into account factors such as pH conditions (e.g., pH gradients), buffer capacity, acceptor sink conditions (physical or chemical), any precipitate of the solute in the donor well, presence of cosolvent in the donor compartment, geometry of the compartments, stirring speeds, filter thickness, porosity, pore size, and tortuosity. 

In PAMPA measurements each well is usually a one-point-in-time (single-timepoint) sample. By contrast, in the conventional multitimepoint Caco-2 assay, the acceptor solution is frequently replaced with fresh buffer solution so that the solution in contact with the membrane contains no more than a few percent of the total sample concentration at any time. This condition can be called a physically maintained sink. Under pseudo-steady state (when a practically linear solute concentration gradient is established in the membrane phase see Chapter 2), lipophilic molecules will distribute into the cell monolayer in accordance with the effective membrane-buffer partition coefficient, even when the acceptor solution contains nearly zero sample concentration (due to the physical sink). If the physical sink is maintained indefinitely, then eventually, all of the sample will be depleted from both the donor and membrane compartments, as the flux approaches zero (Chapter 2). In conventional Caco-2 data analysis, a very simple equation [Eq. (7.10) or (7.11)] is used to calculate the permeability coefficient. But when combinatorial (i.e., lipophilic) compounds are screened, this equation is often invalid, since a considerable portion of the molecules partitions into the membrane phase during the multitimepoint measurements. 

If serum protein or surfactant is added to the acceptor wells, then, in general, p[A l> and P r> are not the same, even under iso-pH conditions. The acceptor-to-donor permeability needs to be solved by performing a separate iso-pH assay, where the serum protein or surfactant is added to the donor side, instead of the acceptor side. The value of Pe is determined, using Eq. (7.20), and used in gradient-pH cases in place of P A /) , as described in the preceding section. The gradient-pH calculation procedure is iterative as well. 

The combination of increased Pe and decreased %R allowed the permeation time to be lowered to 4 h, in comparison to the originally specified time of 15 h [547,550], a considerable improvement for high-throughput applications. The quality of the measurements of the low-permeability molecules did not substantially improve with sink conditions or the reduced assay times. 

Figure 7.35 shows the characteristic log Pe-pH curve for a weak base, phenazo-pyridine (pKa 5.15). With bases, the maximum permeability is realized at high pH values. As in Fig. 7.34, the PAMPA assays were performed under iso-pH conditions (same pH in donor and acceptor wells), using the 2% DOPC in dodecane lipid... 

Accumulation/efflux studies can be performed on different cell systems or membrane vesicle preparations. In the accumulation assays, uptake of a probe over time, typically either fluorescent (e.g. calcein-AM (CAM) [25-27]) or radiolabeled, into the cell or membrane vesicles is measured in the presence or absence of a known P-gp inhibitor. As P-gp transports substrates out of the cells, the inhibition of the protein would result in an increase in the amount of the probe in the cell. Accumulation studies in cells that overexpress P-gp can be compared to those obtained in the parental cell line that does not have as high a level of P-gp expression. The probe in the absence of inhibitors shows lower accumulation in P-gp expressing cells than in P-gp deficient cells. Similarly, probe accumulation is increased under conditions where P-gp is inhibited such that the difference in accumulation in P-gp deficient and overexpressing cells, respectively, becomes smaller. Accumulation assays poorly distinguish substrates and inhibitors of P-gp and, as far as transport assays are concerned, are also influenced by a passive diffusion property of molecules [20]. In contrast to transport assays, both accumulation (i.e. calcein-AM assay) and ATPase assays tend to fail in the identification ofrelatively low permeable compounds as P-gp active compounds [20]. 

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